Adding ROS quenchers to cold K+ cardioplegia reduces superoxide emission during 2-hour global cold cardiac ischemia

Abstract

We reported that the combination of reactive oxygen species (ROS) quenchers Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), catalase, and glutathione (MCG) given before 2 hours cold ischemia better protected cardiac mitochondria against cold ischemia and warm reperfusion (IR)-induced damage than MnTBAP alone. Here, we hypothesize that high K(+) cardioplegia (CP) plus MCG would provide added protection of mitochondrial bioenergetics and cardiac function against IR injury. Using fluorescence spectrophotometry, we monitored redox balance, ie reduced nicotinamide adenine dinucleotide and flavin adenine dinucleotide (NADH/FAD), superoxide (O(2) (•-)), and mitochondrial Ca(2+) (m[Ca(2+)]) in the left ventricular free wall. Guinea pig isolated hearts were perfused with either Krebs Ringer's (KR) solution, CP, or CP + MCG, before and during 27°C perfusion followed immediately by 2 hours of global ischemia at 27°C. Drugs were washed out with KR at the onset of 2 hours 37°C reperfusion. After 120 minutes warm reperfusion, myocardial infarction was lowest in the CP + MCG group and highest in the KR group. Developed left ventricular pressure recovery was similar in CP and CP + MCG and was better than in the KR group. O(2) (•-), m[Ca(2+)], and NADH/FAD were significantly different between the treatment and KR groups. O(2) (•-) was lower in CP + MCG than in the CP group. This study suggests that CP and ROS quenchers act in parallel to improve mitochondrial function and to provide protection against IR injury at 27°C.

Figure 1

Time course of changes in developed LVP (A) and diastolic LVP (B) at baseline, during treatment, hypothermia, cold ischemia, and warm reperfusion for control (n=15), CP (n=16), and CP+MCG (n=14) groups. The arrow indicates where the data point was recorded during treatment at 37°C. The solid horizontal bar indicates the period of treatment at 27°C. The period between the two unbroken vertical lines refers to ischemia. *P < 0.05 values vs. baseline within each group; #P < 0.05 each treatment vs. the control group; ¶P < 0.05, CP+MCG vs. CP alone.

Figure 2

Time course of changes in NADH autofluorescence (A) and FAD autofluorescence (B) at baseline, during treatment, hypothermia, cold ischemia, and warm reperfusion in control (n=5), CP (n=5), and CP+MCG (n=5) groups. The arrow indicates where the data point was recorded during treatment at 37°C. The solid horizontal bar indicates the period of treatment at 27°C. The period between the two unbroken vertical lines refers to ischemia. *P < 0.05 values vs. baseline within each group; #P < 0.05 each treatment vs. the control group; ¶P < 0.05, CP+MCG vs. CP alone.

Figure 3

(A). Time course of changes in superoxide (O₂^•−) signal at baseline, during treatment, hypothermia, cold ischemia, and warm reperfusion in control (n=4), CP (n=4), and CP+MCG (n=4) groups. The arrow indicates where the data point was recorded during treatment at 37°C. The solid horizontal bar indicates the period of treatment at 27°C. The period between the two unbroken vertical lines refers to ischemia. Figure 3(B). Mitochondrial Ca²⁺ levels in control (n=6), CP (n=7), and CP+MCG (n=5) groups at baseline and 60 min of reperfusion. *P < 0.05 values vs. baseline within each group; #P < 0.05 each treatment vs. the control group; ¶P < 0.05, CP+MCG vs. CP alone.

Figure 4

Infarct size as a percentage of total ventricular weight measured after 120 min reperfusion from random hearts in control (n=10), CP (n=8), and CP+MCG (n=6). #P < 0.05 each treatment vs. the control group; ¶P < 0.05, CP+MCG vs. CP alone.